BIBC 100 Problem Set #1 4/11/08 1) Go to the PDB website (Protein Data Bank), and retrieve PDB ID “2cpl”. See the “Images and Visualization” box to the right. The programs KiNG, Jmol, Webmol, etc. allow you to look at the structure of this protein and manipulate it in various ways. You will have to experiment with the various programs and see what they do. I find KiNG and Webmol to be the easiest to understand; Jmol isn’t too bad either. All require Java – I ran these programs in the Safari browser, with the Mac OS X operating system. I expect this exercise to take you about 2 hours to complete. What is this protein? What does it do? cyclophilin A, it is an enzyme (isomerase: peptidyl prolyl cis-trans) Is this protein made up of a single amino acid chain, or multiple chains? How long are the chain(s)? One chain, 164 amino acids Describe the secondary structural composition and any major folds/motifs you see in this protein. eight-strand beta sandwich (beta barrel) motif, flanked by two alpha helices (note that under “sequence details”, the database record states that there are 13 beta strands and 3 alpha helices – and it shows that the extra stands/helix are very small) What secondary structural element is made up of amino acids 30 to 42 in this protein? an alpha helix What is the identity of amino acid 30? Proline What atom is the carbonyl oxygen of amino acid 30 hydrogen-bonded to? the amide hydrogen of E34 2) For the small peptide DENSRREETSCW, what is the overall charge of the peptide at pH 7.4? Each D and E residue is negatively charged at pH 7.4, and each R residue is positively charged, so 4 negative charges and 2 positive charges gives an overall (net) charge of negative two. (Note that the charges on the amino and carboxyl termini counteract one another)3) The molecular mass of serine is 105 Da, and thus the molecular mass of three individual molecules of serine is 315 Da. However, a tripeptide composed of three serine residues has a molecular mass of 279 Da. Why does the peptide have this smaller mass? During the formation of the tripeptide two peptide bonds are formed. Each peptide bond is formed through a dehydration reaction and thus results in the loss of a water molecule, 18 daltons. 315 Da – (2 x 18 Da/H2O) = 279 Da (mass of the tripeptide) 4) What would be the approximate molecular weight of a 600 a.a. protein? (Hint: see the discussion of the range of sizes of peptides and proteins in Chapter 3) 600 a.a. X 110 daltons avg mass/ a.a. = 66,000 or 66 kD 5) Plot the following 10-residue peptide as an alpha-helix on the helical wheel (see slide 12, Lecture 3, from B and T figure 2-4). Make sure to mark the details such as N-terminus, C-terminus, the distance/angle between each residue, hydrophobic region/polar region. N-Terminus –M-T-R-A-A-S-L-I-Q-S – C-Terminus6) Go to the NCBI website, and BLAST (protein BLAST) this sequence: MAPWMHLLTVLALIALWGPNSVQAYSSQHLCGSQLVEALFMTCGRSGFYRPHDKKELEDLQVEQAELGLDAGGLQPSALDMILQKRGIVDQCCNNICTYNQLQNYCNVP What is this protein? What type of organism has this protein? Insulin precursor protein, or Preproinsulin. This particular preproinsulin sequence is almost identical to the preproinsulin from Octodon degus, a rodent (this rodent is very gerbil-like). I found this out by clicking on the link next to the most similar sequence found by BLAST. An “E-value or Expect value” is a statistical measure of similarity between proteins. One of your BLAST hits has an expect value of 2e-27 (very similar). The BLAST program shows an alignment between your query sequence and this protein. How many amino acids are identical to one another in the alignment? How many are similar to one another? The alignment with the Rattus losea preproinsulin1 shows an expect value of 2e-27. 55% of the amino acids are identical in this alignment (61/110), another 14% (15/110) are similar to one
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